Compounds, compositions and methods of treating or preventing acute lung injury

ABSTRACT

The invention includes methods of preventing or treating acute lung injury using a MAP3K2/MAP3K3 inhibitor. The invention further comprises compositions, and kits comprising compositions useful within the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national phase application from,and claims priority to, International Application No. PCT/US2018/027980,filed Apr. 17, 2018, and published under PCT Article 21(2) in English,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 62/486,232, filed Apr. 17, 2017, all of whichapplications are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL135805 awardedby National Institutes of Health. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The incidence of acute lung injury (ALI) and its more severe form, acuterespiratory distress syndrome (ARDS), is reported to be around 200,000per year in the US with a mortality rate of around 40%. The diseases arethe manifestations of an inflammatory response of the lung to direct orindirect insults, and are characterized by severe hypoxemia and asubstantial reduction in pulmonary compliance due to diffuse alveolardamage, neutrophilic inflammation, and protein-rich edema in the lungs.Care of these conditions is largely dependent on supportive measures.There is currently a lack of effective pharmacological interventions.Pharmacological therapies that have been tested in patients withALI/ARDS failed to reduce mortality. There is thus a clear unmet medicalneed for therapeutic intervention of this disease.

One of the hallmarks of ALI is abundant presence of neutrophils in thelungs. Neutrophils are the most abundant leukocytes in humancirculation, playing important roles in innate immunity againstmicrobial infections and also contributing to inflammation-relatedtissue damages. During the inflammation, neutrophils are recruited tothe sites of injury and infection from circulation through a multi-stepprocess, which includes rolling and firm adhesion on endothelial cells,intravascular crawling, diapedesis, and extravascular chemotaxis. Onceat the sites, neutrophils perform a number of tasks includingphagocytosis, release of preformed granule enzymes, and production ofreactive oxygen species (ROS). Evidence has clearly linked neutrophilsto the pathogenesis of ALI/ARDS. Although crossing of the alveolarepithelium by neutrophils does not directly cause an increase in lungepithelial permeability, neutrophils play important roles in pulmonaryedema with the underlying mechanisms that remain incompletelyunderstood.

While neutrophil extracellular traps and granule enzymes such asneutrophil elastase contribute to the pathology of ALI, including lungedema, any role of ROS in ALI/ARDS is still debatable. Neutrophilsproduce ROS primarily through the phagocyte NADPH oxidase, which is amember of the NOX family. It consists of four cytosolic components(p47^(phox), p67^(phox), p40^(phox), and Rac) and two membrane subunits(gp91^(phox)/NOX2 and p22^(phox)). When the cells are activated bystimuli such as chemo-attractants, the cytosolic components arerecruited to the membrane components to form the active holoenzyme toproduce ROS. One of the key activation events is the phosphorylation ofthe cytosolic p47^(phox) subunit by protein kinases including PKC. Thephosphorylation disrupts auto-inhibitory intramolecular interactioninvolving the internal SH3 domains, leading to its interaction withp22^(phox), required for the activation of the NADPH oxidase.

MAP3K2 and MAP3K3 are two highly conserved members of the MEK kinase(MEKK) subgroup of the MAP3K superfamily. They contain a kinase domainin the C terminus and a PB1 domain near the N terminus. The kinasedomains of MAP3K2 and MAP3K3 share 94% sequence identity, and these twokinases are expected to share substrates. Transient expression of thekinases in vitro leads to their auto-activation and activation of ERK1and ERK2, p38, JNK, and ERK5. In mice, these kinases are involved incardiovascular development, lymphocyte differentiation and NF-kappaBregulation. However, their roles in primary myeloid cell biology or ALIhave not been investigated.

There is a need in the art to identify novel therapeutic treatments thatcan be used to treat or prevent ALI/ARDS in patients afflicted withthose diseases. The present invention addresses and meets this need.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of treating or preventing acute lunginjury (ALI) in a subject in need thereof. The invention furtherprovides a method of treating or preventing lung fibrosis in a subjectin need thereof. The invention further comprises a kit comprising acompound or composition useful within the methods of the invention.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of pazopanib, or a salt orsolvate thereof. In other embodiments, the administration route is oral.In other embodiments, the administration route is parenteral. In yetother embodiments, the administration route is nasal. In yet otherembodiments, the administration route is inhalational. In yet otherembodiments, the administration route is intratracheal. In yet otherembodiments, the administration route is intrapulmonary. In yet otherembodiments, the administration route is intrabronchial. In yet otherembodiments, the administration route is selected from the groupconsisting of oral, parenteral, nasal, inhalational, intratracheal,intrapulmonary, and intrabronchial. In yet other embodiments, theadministration route is selected from the group consisting of nasal,inhalational, intratracheal, intrapulmonary, and intrabronchial. In yetother embodiments, the administration is done using a nebulizer.

In certain embodiments, the subject is in an intensive care unit (ICU)or emergency room (ER). In other embodiments, the acute lung injury isacute respiratory distress syndrome (ARDS).

In certain embodiments, the subject is further administered at least oneadditional agent and/or therapy that treats, prevents or reduces thesymptoms of the acute lung injury. In other embodiments, the subject isfurther administered at least one additional agent and/or therapy thattreats, prevents or reduces the symptoms of the lung fibrosis.

In certain embodiments, the pazopanib, or a salt or solvate thereof, isadministered to the subject at a frequency selected from the groupconsisting of about three times a day, about twice a day, about once aday, about every other day, about every third day, about every fourthday, about every fifth day, about every sixth day and about once a week.In other embodiments, the pazopanib, or a salt or solvate thereof, isformulated as a dry powder blend.

In certain embodiments, administration of the pazopanib, or a salt orsolvate thereof, to the subject does not cause at least one significantadverse reaction, side effect and/or toxicity associated withoral/systemic administration of the pazopanib, or a salt or solvatethereof, to a subject suffering from cancer. In other embodiments, theat least one adverse reaction, side effect and/or toxicity is selectedfrom the group consisting of hepatotoxicity, prolonged QT intervals andtorsades de pointes, hemorrhagic event, decrease or hampering ofcoagulation, arterial thrombotic event, gastrointestinal perforation orfistula, hypertension, hypothyroidism, proteinuria, diarrhea, hair colorchanges, nausea, anorexia, and vomiting.

In certain embodiments, the subject is dosed with an amount ofpazopanib, or a salt or solvate thereof, that is lower than the amountof pazopanib, or a salt or solvate thereof, with which a subjectsuffering from cancer is dosed orally/systemically for cancer treatment.

In certain embodiments, the subject is a mammal. In other embodiments,the mammal is a human.

In certain embodiments, the kit comprises pazopanib, or a salt orsolvate thereof. In other embodiments, the kit comprises an applicator.In yet other embodiments, the kit comprises an instructional materialfor use thereof. In yet other embodiments, the kit comprises at leastone additional agent that treats, prevents or reduces the symptoms ofthe acute lung injury and/or lung fibrosis. In yet other embodiments,the instructional material comprises instructions for treating orpreventing acute lung injury and/or lung fibrosis in a subject.

The invention further provides a method of evaluating efficacy of a drugin treating ALI. In certain embodiments, the method comprises contactinga neutrophil with the drug and measuring neutrophil ROS productionlevels after the contacting. In other embodiments, if the neutrophil ROSproduction levels increase after the contacting, the drug is efficaciousin treating ALI.

The invention further provides a method of evaluating efficacy of a drugin treating a subject suffering from ALI. In certain embodiments, themethod comprises measuring neutrophil ROS production levels in thesubject after being administered the drug. In other embodiments, if theneutrophil ROS production levels in the subject after being administeredthe drug are higher than the neutrophil ROS production levels in thesubject before being administered the drug, the drug is efficacious intreating ALI in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention,specific embodiments are shown in the drawings. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities of the embodiments shown in the drawings.

FIGS. 1A-1H illustrate loss of MAP3K2 in hematopoietic cells and MAP3K3in myeloid cells ameliorates LPS-induced lung injury. FIG. 1Aillustrates loss of MAP3K2 and 3 proteins in HS-DKO neutrophils. FIG. 1Billustrates reduced pulmonary permeability in HS-DKO mice. HS-DKO andcontrol WT mice were treated with LPS via intranasal route. Lungpermeability to FITC-labeled albumin was determined by measuring thefluorescence of BAL 24 hours after injury induction. FIG. 1F illustratesreduced pulmonary permeability in HS-DKO mice. HS-DKO and control WTmice were treated with HCl via oral-tracheal intubation. Lungpermeability to FITC-labeled albumin was determined by measuring thefluorescence of BAL 6 hours after injury induction. Data are presentedas mean±sem (Student t-Test, *p<0.05, n=8). FIGS. 1C-1D & 1G illustraterepresentative histology of lung samples from FIG. 1B and FIG. 1F,respectively. Br, bronchus; V, blood vessel; *, edema. FIG. 1Eillustrates HS-DKO mice show extended survival by LPS-induced lunginjury, and FIG. 1H illustrates HS-DKO mice show extended survival byHCl-induced lung injury. The mice were treated as in FIG. 1B and FIG.1F, and observed for survival (the Mantel-Cox Log-Rank test).

FIGS. 2A-2K illustrate the finding that MAP3K2/3-null neutrophil shownormal chemotaxis, endothelial cell adhesion, integrin expression andactivation, infiltration and degranulation. FIGS. 2A-2D illustrate thefinding that MAP3K2/3-null neutrophil show normal chemotaxis.Representative cell migration traces from a Dunn chamber chemotaxisassay are shown in FIGS. 2A-2B. The translocation and directionalityparameters for how fast the cells move and how well they follow thechemoattractant gradient are shown in FIGS. 2C-2D. n=50. FIG. 2Eillustrates adhesion of neutrophils to endothelial cells under shearflow. n=3. FIGS. 2F-2G illustrate cell surface expression of LFA-1 andMAC-1 integrins on neutrophils. n=3. FIG. 2H illustrates binding ofneutrophils to ICAM-1, which reflects the avidity of integrins onneutrophil upon activation. n=3. FIG. 2I illustrates infiltration ofneutrophils into inflamed peritonea. n=5. FIGS. 2J-2K illustratesrelease of MMP and MPO from neutrophil granules upon stimulation. n=3.

FIGS. 3A-3E illustrate the finding that MAP3K2/3 inhibits ROS releasefrom neutrophils dependently of kinase activity. FIG. 3A illustrates thefinding that loss of MAP3K2/3 increases ROS release from neutrophils.Representative ROS measurement traces are shown in the left panel,whereas ROS amounts calculated from the areas under the traces from morethan five mice are summarized in the right panel (data are presented asmean±sem, Student t-Test, *p<0.05, n=3). The experiments were repeatedat least 3 times. FIG. 3B illustrates the finding that expression of WTMAP3K3, but not its kinase dead mutant, suppresses ROS production in DKOneutrophils. Neutrophils were transiently transfected with plasmids forGFP, MAK3K3-GFP, MAP3K3 kinase dead (KD) or PB1 domain-deletion mutantfused with GFP. GFP-positive cells were sorted the next day and used forROS release assay. The expression of MAP3K3 and its mutants weredetected by Western analysis. Data are presented as mean±sem (Studentt-Test, *p<0.05, n=3). FIGS. 3C-3E illustrate the finding thatsuperoxide scavenger BHA abrogates the difference between HS-DKO andcontrol WT mice in LPS-induced lung injury. Mice under diet containingBHA were subjected to LPS-induced lung injury. n=5.

FIGS. 4A-4G illustrate the finding that MAP3K3 phosphorylates S208 ofp47phox to inhibit NADPH oxidase activity. FIG. 4A illustrates thefinding that MAP3K3 phosphorylates p47phox. In vitro kinase assay wasperformed using recombinant MAPK3K3 and NADPH oxidase subunitsimmunoprecipitated from HEK293 cells. The NADPH oxidase subunits weretransiently expressed with an HA-tag, and anti-HA antibody was used forimmunoprecipitation. FIG. 4B illustrates the finding that MAP3K3phosphorylates S208 of p47phox. In vitro kinase assay was performedusing recombinant MAP3K3 and GST-fused fragment (p47SH3) of wild type(WT) or S208E mutated (SE) p47phox (residues 151-286) that contains thetwo SH3 domains. The quantification of the phosphorylation was done by aphosphoimager. FIG. 4C illustrates the finding that phosphomimeticmutation of Ser-208 of p47phox leads to reduced activity in thereconstituted ROS production assay. COS-7 cells were cotransfected withplasmids for p22phox, p67phox, and p97phox together with WT p47phox orits S208A (SA) or S208E (SE) mutant. The PMA-induced ROS production areshown. Data are presented as mean±sem (Student t-Test, *p<0.05, n=5).FIG. 4D illustrates the finding that WT p47phox, but not its S208Amutant, is inhibited by MAP3K3. COS-7 cells were cotransfected withplasmids for p22phox, p67phox, and p97phox together with WT p47phox(left panel) or its S208A (right panel) mutant in the presence orabsence of MAP3K3. The PMA-induced ROS production are shown. Data arepresented as mean±sem (Student t-Test, *p<0.05, n=5). FIG. 4Eillustrates the finding that phosphomimetic mutation of Ser-208 ofp47phox impairs the interaction with p22phox. GST pull-down assay wasperformed with recombinant GST-p47SH3 carrying a substitution of Ala orGlu for Ser-208 and MBP-fused C-terminus (residues 96-164) of p22phox(p22C). Western analysis was used for detection of the proteins. FIG. 4Fillustrates the finding that phosphorylation of Ser-208 of p47phox isstimulated by fMLP. Neutrophils were stimulated with fMLP (1 μM) forvarying durations, followed by Western analysis. FIG. 4G illustrates thefinding that FMLP-stimulated p47phox phosphorylation depends onMAP3K2/3.

FIGS. 4H-4K illustrate the finding that pazopanib inhibitsphosphorylation of p47phox by MEKK2 or 3 in an in vitro kinase assay.Recombinant p47phox prepared from an E. coli expression system wereincubated with recombinant MAP3K2 or 3 in the presence of ATP for 30 minat 37° C. The proteins were analysis by Western blotting. The IC₅₀ forinhibition of phosphorylation of p47 by MAP3K2 is around 20 nM, whereasthe IC₅₀ for MAP3K3 is around 10 nM. These numbers are far lower thanthe previously reported values for the effects of pazopanib on ATPbinding or phosphorylation of MEK5 (FIGS. 4J-4K) by MAP3K2, which werehigher than 500 nM) See Ahmad, et al., 2013, J. Biomol. Screen. 18:388.

FIGS. 5A-5F illustrate the finding that pazopanib inhibits MAP3K2/3 andinduces phenotypes similar to those of genetic MAP3K2/3 inactivation.FIG. 5A illustrates the finding that pazopanib inhibits phosphorylationof Ser-208 of p47phox. Neutrophils were pretreated with pazopanib (pazo)for 10 min before stimulation by fMLP (1 μM), followed by Westernanalysis. FIGS. 5B-5C illustrate the finding that pazopanib increasesROS release from neutrophils depending on MAP3K2/3. Neutrophils werepretreated with pazopanib (20 nM in FIG. 5C) for 10 min beforestimulation by fMLP (1 μM) and ROS measurement. Data are presented asmean±sem (Student t-Test, *p<0.05, n=3). FIGS. 5D-5E illustrate thefinding that pazopanib treatment attenuates LPS-induced lung injury.Mice (C57B1 female) were treated with 60 mg/Kg/day pazopanib via gavagetwo day before lung injury induction by LPS. One day after lung injuryinduction, lung permeability (FIG. 5D) and histology (FIG. 5E) wereexamined. The experiment was repeated twice with similar outcomes. Datafrom one experiment are presented as mean±sem (Student t-Test, *p<0.05,n=5). FIG. 5F illustrates the finding that pazopanib treatment reducesmortality of mice with LPS-induced lung injury. The C57B1 mice weretreated as above and their survival was analyzed by the Mantel-CoxLog-Rank test.

FIG. 6, comprising Panels A-D, illustrates the finding that AKT ishyperactivated in LPS-inured lungs of HS-DKO. Sections from LPS-inuredlungs were stained for phospho-AKT and CD31 (Panels A-B) or smoothmuscle actin (SMA; Panels C-D). Phospho-AKT staining is elevated inareas co-stained by CD31 (compare closed triangles) and SMA stainingadjacent to blood vessels (V) (compared open triangles). By contrast,phospho-AKT staining at brachial walls (solid arrows) and brachialsmooth muscle cells stained by SMA next to brachial wall (open arrows)remains the same between HS-DKO and WT samples. Images for CD31 and SMAstaining alone are shown in FIGS. 13A-13F.

FIGS. 7A-7D illustrate the finding that neutrophil lacking MAP3K2/3increase AKT activation in endothelial cells via H₂O₂. FIG. 7Aillustrate a non-limiting model that describes how MAP3K2/3 inhibitionleads to the increase in ROS production from neutrophils and AKThyperactivation in endothelial cells as well as pericytes. Withoutwishing to be limited by any theory, hyperactivation of AKT leads toimproved vascular integrity and reduced permeability, thus the healthierlungs during ALI. FIGS. 7B-7C illustrate the finding that co-culture ofMAP3K2/3-deficienct neutrophils (DKO) causes greater AKT phosphorylationcompared to that of WT neutrophils, and this difference in AKTphosphorylation is abrogated by the presence of catalase (Cat), but notsuperoxide dismutase (SOD). FIG. 7D illustrates TEER measurement ofmouse lung endothelial cells co-cultured with WT or DKO neutrophils inthe presence or absence of SOD. The arrow indicates the time point atwhich neutrophils were added.

FIGS. 8A-8E illustrate the finding that loss of MAP3K2 in hematopoieticcells and MAP3K3 in myeloid cells does not affect the number ofinfiltrated myeloid cells or contents of cytokines in BALF ofLPS-injured lungs. FIG. 8A illustrate a validation of LPS-induced lunginjury model. Mice were treated with LPS via intranasal route. Lungpermeability to FITC-labeled albumin was determined by measuring thefluorescence in BALF 24 hours after injury induction. Data are presentedas mean±sem (Student t-Test, n=4). n=5. FIGS. 8B-8E illustrate how wholelung from mice described in FIGS. 1A-1E were analyzed by flow cytometry(FIGS. 8A-8D) and by ELISA (FIG. 8E).

FIGS. 8F-8J illustrate the finding that loss of MAP3K2 in hematopoieticcells and MAP3K3 in myeloid cells does not affect the number ofinfiltrated myeloid cells or contents of cytokines in BALF ofHCl-injured lungs. FIG. 8F illustrates a schematic of HCl-induced lunginjury model. FIGS. 8H-8J illustrate how whole lung were analyzed byflow cytometry (FIGS. 8G-8I) and by ELISA (FIG. 8J).

FIGS. 9A-9B illustrate ROS release from neutrophils lacking MAP3K2 orMAP3K3 upon stimulation of fMLP.

FIGS. 10A-10D illustrate validation of the reconstituted ROS productionsystem in COS-7 cells. FIGS. 10A-10B: COS-7 cells were transfected withplasmids for NANPH oxidase subunits as indicated in the figures andtreated with and without PMA. ROS production and protein expression weredetermined. FIG. 10C illustrates the finding that WT MAP3K3, but not itskinase dead mutant, can inhibit ROS production in the reconstitutedCOS-7 system. FIG. 10D illustrates a non-limiting schematic model thatdepicts how MAP3K2/3 suppresses ROS production.

FIG. 11 illustrates validation of anti-phospho-S208 p47^(phox) antibody.HEK293 cells were cotransfected with WT or kinase dead MAP3K3 togetherwith WT or S208A p47^(phox). Western analysis was performed the nextday.

FIGS. 12A-12K illustrate the finding that LPS induced lung injury byincreasing pulmonary permeability. FIGS. 12A-12B illustrate the findingthat pazopanib inhibits MEKK3 and increases ROS production from humanneutrophils. Human neutrophils were stimulated with fMLP (100 nM) in thepresence and absence of 20 nM pazopanib. Data in FIG. 12B are presentedas mean±sem (*, P<0.05, student t-test; n=5). FIGS. 12C-12D illustratethe finding that BHA abrogates pazopanib's effect on lung permeability.HS-DKO Mice were fed on regular chew or chew containing BHA and treatedwith 60 mg/Kg/day pazopanib via gavage starting two days before lunginjury induction by LPS. One day after lung injury induction, lungpermeability was determined. Data in FIG. 12C are presented as mean±sem(n=5). Representative histology is shown in FIG. 12D. FIG. 12Eillustrates the finding that therapeutic treatment of pazopanib reducesmortality of mice with LPS-induced lung injury. Mice (C57B1 female, 8weeks) were treated with 1.5 mg/Kg pazopanib via intra-nasal 24 h afterlung injury induction by LPS (80 μg/g, 32 mg/ml) and their survival wasanalyzed by the Mantel-Cox Log-Rank test. FIGS. 12F-12G illustrates thefinding that pazopanib treatment attenuates HCl-induced lung injury.Mice (C57B1 female, 8 weeks) were treated with 1.5 mg/Kg pazopanib viaintra-nasal 1 h after lung injury induction by HCl (0.05 M, 2.5 μl/g).Six hours after lung injury induction, lung permeability (FIG. 12D) andhistology (FIG. 12E) were examined. The experiment was repeated twicewith similar outcomes. Data from one experiment are presented asmean±sem (Student t-Test, *p<0.05, n=5). FIG. 12H illustrates thefinding that pazopanib treatment reduces mortality of mice withLPS-induced lung injury. Mice (C57B1 female, 8 weeks) were treated with2.5 mg/Kg pazopanib via intra-nasal 1 h after lung injury induction byHCl (0.1 M, 2.5 μl/g) and their survival was analyzed by the Mantel-CoxLog-Rank test. FIG. 12I-12K illustrates the finding that preventativetreatment of pazopanib reduces lung permeability and mortality of micewith HCl-induced lung injury. FIG. 12J illustrates the finding thatpazopanib pretreatment attenuates HCl-induced lung injury. Mice (C57B1female, 8 weeks) were treated with 1.5 mg/Kg pazopanib via intra-nasal0.5 hour before lung injury induction by HCl (0.05 M, 2.5 μl/g). Sixhours after lung injury induction, lung permeability were examined. Theexperiment was repeated twice with similar outcomes. Data from oneexperiment are presented as mean±sem (Student t-Test, *p<0.05, n=5).FIG. 12K illustrates the finding that pazopanib pretreatment reducesmortality of mice with HCl-induced lung injury. Mice (C57B1 female, 8weeks) were treated with 1.5 mg/Kg pazopanib via intra-nasal 0.5 hourbefore lung injury induction by HCl (0.1 M, 2.5 μl/g) and their survivalwas analyzed by the Mantel-Cox Log-Rank test.

FIGS. 13A-13F illustrate hyperactivation of phospho-AKT by MAP3K2/3inactivation. FIG. 13A illustrates increases in phosphorylation of AKTat S473 in the protein extracts from LPS-induced lungs of HS-DKO mice.FIGS. 13B-13C illustrate supplementary images of CD31 and SMA stainingalone of LPS-inured lung sections for FIG. 6, Panels A-D. FIGS. 13D-13Eillustrate an effect of pazopanib on AKT phosphorylation in lung samplesfrom WT and HS-DKO mice. FIG. 13F illustrates the finding thatAKT-inhibitor MK-2206 abrogates the effect of pazopanib on permeabilityin LPS-injured lungs. Mice were treated with MK-2206 (10 mg/Kg) in thepresence or absence of 60 mg/Kg/day pazopanib via gavage starting twodays before lung injury induction by LPS. n=5.

FIGS. 14A-14D illustrate activation of Rac1 by MAP3K2/3 inactivation.FIGS. 14A and 14B illustrates Rac1 activation in the protein extractsfrom H2O2-induced mouse lung endothelial cells (MLEC). FIGS. 14C-14Dillustrates Rac1 activation in MLEC which were co-cultured with fMLPinduced MAP3K2/3 deficient neutrophils.

FIG. 15 illustrates the finding that Avastin, a VEGF inhibitor, haslimited effects on HCl-induced acute lung injury.

FIGS. 16A-16E illustrate the finding that pazopanib treatment attenuatesHCl (FIGS. 16A-16C) or LPS-induced (FIGS. 16D-16E) lung injury in atherapeutic modality. FIGS. 16A and 16D illustrate treatment results forWT and p47-HKO mice. FIGS. 16B-16C illustrate the effect of pazopanib onpermeability, whereas FIGS. 16D-16E illustrate beneficial effects ofpazopanib on survival. FIGS. 16A, 16B and 16D illustrate the findingthat loss of p47^(phox), a key element for ROS generation, exacerbatelung permeability (FIGS. 16A-16B) and decrease survival rate (FIG. 16D)in HCl-induced lung injury. Loss of p47^(phox) abrogated the therapeuticeffect of pazopanib indicated by HCl-induced lung permeability (FIG.16C) and survival rate (FIG. 16E).

FIG. 17 is a graph illustrating the result that imatinib(4-[(4-methylpiperazin-1-yl)methyl]-N-(4-methyl-3-{[4-(pyridin-3-yl)pyrimidin-2-yl]amino}phenyl)benzamide,or GLEEVEC®) treatment does not attenuate HCl-induced lung injury. Mice(C57B1 female, 8 weeks) were treated with 1.5 mg/Kg Imatinib orpazopanib intra-nasally 1 h after lung injury induction by HCl. Sixhours after lung injury induction, lung permeability (D) and histology(E) were examined. Data are presented as mean±sem (Student t-Test).Imatinib has a trend of aggravation of the injury.

FIG. 18 is a bar graph illustrating the result that pazopanib inhibitsbleomycin-induced lung fibrosis. Mice (C57B1 female, 8 weeks) weretreated with 0.05 unit bleomycin once. One week later, the mice weregiven orally 60 mg/kg pazopanib for five days, and the lung fibrosis wasdetermined by measuring the levels of hydroxyproline.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in part to the unexpected discovery thatincreased reactive oxygen species (ROS) production from neutrophils byMAP3K2 and/or MAP3K3 inhibition protects lung during acute injury. ROSis generally believed to exacerbate tissue injuries. However, asdemonstrated herein, moderate elevation of NADPH oxidase-derived ROS inneutrophils ameliorates acute lung injury (ALI) manifestations andreduces mortality in mice. MAP3K2 and MAP3K3 were herein identified asbeing novel negative regulators of neutrophil NADPH oxidase byphosphorylating p47^(phox) at Serine 208. Neutrophils lacking MAP3K3 andits homolog MAP3K2 produce a greater amount of ROS, while showing normalchemotaxis, adhesion to endothelial cells, infiltration, anddegranulation. Genetic loss of MAP3K3 in myeloid cells and MAP3K2 inhematopoietic cells was found to protect mice from pulmonary edema andmortality in a mouse ALI model, accompanied by enhanced AKT activationin the lung vasculature. These phenotypes can be recapitulated by aMAP3K2/3 inhibitor pazopanib.

Thus, these present study sheds new light on the role of ROS in ALI andreveals a previously unknown mechanism for regulation of ROS production.Further, it provides a potential target and agent for therapeuticintervention of ALI, a life-threatening disease that currently lackspharmacological treatment. In a non-limiting aspect, these resultssupport the therapeutic potential of aerosolized administration ofpazopanib to subjects suffering from ALI. In certain embodiments,targeted administration of pazopanib within injured lung attenuate orcompletely resolve ALI, for example by treating, reversing orameliorating diffuse alveolar damage and/or edema.

The present invention provides a method of treating or preventing lungfibrosis and/or acute lung injury in a subject, comprising administeringto the subject a therapeutically effective amount of pazopanib or a saltor solvate thereof. In certain embodiments, the pazopanib, or salt orsolvate thereof, is directly delivered into the lung using an inhaler,for example. This allows for effective delivery of an optimal drug dosewithin areas of affected lung, maximizing its therapeutic effects andminimizing potential side effects arising from systemic administration.In certain embodiments, localized delivery of pazopanib minimizes anypossible side effects of increase in ROS production in non-lung tissues.

As discussed herein, a previously unknown function for protein kinasesMAP3K2 and 3 in negative regulation of phagocytic NADPH oxidase wasidentified. These kinases phosphorylate Ser-208 of p47^(phox). Thisphosphorylation, in contrast to previously known phosphorylation sitesin p47^(phox), prevents p47^(phox) interaction with p22^(phox) and leadsto inhibition of the NADPH oxidase activity (FIG. 10D). As expected,either the genetic loss of MAP3K2/3 or their pharmacological inhibitionresulted in increased ROS production. The increased ROS protected micefrom LPS induced ALI.

The present results indicate that pharmacological induction of increasedROS can be protective in a disease model. Most of the attention has beengiven to the detrimental effects of ROS, as excessive amounts of ROS cancause damage to lipids, proteins, and DNA. At the time of the inventionit was unknown whether an increase in ROS production would be effectivein curbing inflammatory responses and provide beneficial therapeuticeffects, in particular in a clinically practical manner. The presentstudies demonstrate that genetic or pharmacological inhibition of MAP3K2and MAP3K3 leads to increases in ROS production in neutrophils andattenuates lung injury in mice, the latter of which depends on ROS.Pazopanib, an FDA-approved small molecular drug, which inhibitsMAP3K2/3, elevates ROS in both human and mouse neutrophils andalleviates lung injury phenotypes in mice, provides a clinicallyfeasible way to achieve the therapeutic benefits. Without wishing to belimited by any theory, once it being released outside cells, ROS can beconverted to H₂O₂ as superoxide dismutase (SOD) is abundantly present inlung tissues, and H₂O₂ has at moderate levels a protective role inpulmonary vasculatures integrity, leading to reduction in permeabilityand edema during injury. In certain non-limiting embodiments, increasedROS generation either with the genetic loss of MAP3K2/3 or with theirpharmacologic inhibition by pazopanib represents an optimal situationwhere ROS was sufficient to activate protective AKT phosphorylation butnot high enough to cause irreversible damage. In certain embodiments,increased ROS production in neutrophils can be used as a readout forefficacy of drugs being used to treat ALI in a subject

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

As used herein, the articles “a” and “an” are used to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, “about,” when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, the phrase “acute lung injury” or “ALI” refers to asyndrome consisting of acute hypoxemic respiratory failure withbilateral pulmonary infiltrates, which is associated with both pulmonaryand nonpulmonary risk factors and that is not primarily due to leftatrial hypertension.

As used herein, the phrase “acute respiratory distress syndrome” or“ARDS” refers to a subtype of acute lung injury characterized by moresevere hypoxemia.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

In one aspect, the terms “co-administered” and “co-administration” asrelating to a subject refer to administering to the subject a compoundof the invention or salt thereof along with a compound that may alsotreat the disorders or diseases contemplated within the invention. Incertain embodiments, the co-administered compounds are administeredseparately, or in any kind of combination as part of a singletherapeutic approach. The co-administered compound may be formulated inany kind of combinations as mixtures of solids and liquids under avariety of solid, gel, and liquid formulations, and as a solution.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient orsubject. Multiple techniques of administering a compound exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, nasal, pulmonary and topical administration.

A “disease” as used herein is a state of health of an animal wherein theanimal cannot maintain homeostasis, and wherein if the disease is notameliorated then the animal's health continues to deteriorate.

A “disorder” as used herein in an animal is a state of health in whichthe animal is able to maintain homeostasis, but in which the animal'sstate of health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionthat can be used to communicate the usefulness of the composition and/orcompound of the invention in a kit. The instructional material of thekit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container that contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

The terms “patient,” “subject” or “individual” are used interchangeablyherein, and refer to any animal, or cells thereof whether in vitro or insitu, amenable to the methods described herein. In a non-limitingembodiment, the patient, subject or individual is a human.

As used herein, the term “pazopanib” refers to5-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)-2-methylbenzenesulfonamide,or a salt and/or solvate thereof:

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

As used herein, “pharmaceutically acceptable carrier” also includes anyand all coatings, antibacterial and antifungal agents, and absorptiondelaying agents, and the like that are compatible with the activity ofthe compound useful within the invention, and are physiologicallyacceptable to the patient. Supplementary active compounds may also beincorporated into the compositions.

The “pharmaceutically acceptable carrier” may further include apharmaceutically acceptable salt of the compound useful within theinvention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

The term “prevent,” “preventing” or “prevention,” as used herein, meansavoiding or delaying the onset of symptoms associated with a disease orcondition in a subject that has not developed such symptoms at the timethe administering of an agent or compound commences.

As used herein, the term “ROS” refers to reactive oxygen species.Non-limiting examples of ROS are peroxide, superoxide, hydroxyl radical,and singlet oxygen.

The term “salt” embraces addition salts of free acids and/or basis thatare useful within the methods of the invention. The term“pharmaceutically acceptable salt” refers to salts that possess toxicityprofiles within a range that affords utility in pharmaceuticalapplications. Pharmaceutically unacceptable salts may nonethelesspossess properties such as high crystallinity, which have utility in thepractice of the present invention, such as for example utility inprocess of synthesis, purification or formulation of compounds and/orcompositions useful within the methods of the invention. Suitablepharmaceutically acceptable acid addition salts may be prepared from aninorganic acid or from an organic acid. Examples of inorganic acidsinclude hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric(including sulfate and hydrogen sulfate), and phosphoric acids(including hydrogen phosphate and dihydrogen phosphate). Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,araliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which include formic, acetic, propionic, succinic,glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic,glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic,p-toluenesulfonic, trifluoromethanesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable pharmaceutically acceptablebase addition salts of compounds and/or compositions of the inventioninclude, for example, metallic salts including alkali metal, alkalineearth metal and transition metal salts such as, for example, calcium,magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptablebase addition salts also include organic salts made from basic aminessuch as, for example, N,N′-dibenzylethylene-diamine, chloroprocaine,choline, diethanolamine, ethylenediamine, meglumine (also known asN-methylglucamine) and procaine. All of these salts may be prepared fromthe corresponding compound by reacting, for example, the appropriateacid or base with the compound and/or composition.

As used herein, a “solvate” of a compound refers to the entity formed byassociation of the compound with one or more solvent molecules. Solvatesinclude water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) oralcohol (e.g., ethanol) solvates, acetates and the like. In certainembodiments, the compounds described herein exist in solvated forms withsolvents such as water, and ethanol. In other embodiments, the compoundsdescribed herein exist in unsolvated form.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has a condition contemplatedherein, a symptom of a condition contemplated herein or the potential todevelop a condition contemplated herein, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect acondition contemplated herein, the symptoms of a condition contemplatedherein or the potential to develop a condition contemplated herein. Suchtreatments may be specifically tailored or modified, based on knowledgeobtained from the field of pharmacogenomics.

By the term “specifically bind” or “specifically binds,” as used herein,is meant that a first molecule preferentially binds to a second molecule(e.g., a particular receptor or enzyme), but does not necessarily bindonly to that second molecule.

The following non-limiting abbreviations are used herein: ALI, acutelung injury; ARDS, acute respiratory distress syndrome; BSA, bovineserum albumin; DMEM, Dulbecco's Modified Eagle Medium; fMLP,N-Formyl-L-methionyl-L-leucyl-L-phenylalanine; MSS, Hanks balanced salt;HRP, horse radish peroxidase; LPS, lipopolysaccharide; MAP3K2 or MEKK2,mitogen-activated protein kinase kinase kinase 2; MAP3K3 or MEKK3,mitogen-activated protein kinase kinase kinase 3; MEK, mitogen-activatedprotein kinase kinase; MEKK, MEK kinase; PBS, phosphate buffered saline;PFA, paraformaldehyde; PMA, phorbol 12-myristate 13-acetate; RBC, redblood cell; ROS, reactive oxygen species; TG, thioglycolate.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.1, 5.3, 5.5, and6. This applies regardless of the breadth of the range.

Compounds and Compositions

In certain embodiments, pazopanib, or a salt or solvate thereof, isuseful within the methods of the invention. In other embodiments,compounds and/or compositions useful within the invention are recited inU.S. Pat. Nos. 7,105,530; 7,262,203; 7,858,626; and 8,114,885; all ofwhich are incorporated herein in their entireties by reference.Compositions comprising pazopanib, or a salt or solvate thereof, arealso contemplated within the invention.

Methods

The invention includes a method of preventing or treating acute lunginjury in a subject in need thereof. The invention includes a method ofpreventing or treating lung fibrosis in a subject in need thereof.

In certain embodiments, the method comprises administering to thesubject therapeutically effective amounts of pazopanib, or a salt orsolvate thereof. In other embodiments, the administration route is oral.In other embodiments, the administration route is parenteral. In yetother embodiments, the administration route is nasal. In yet otherembodiments, the administration route is inhalational. In yet otherembodiments, the administration route is intratracheal. In yet otherembodiments, the administration route is intrapulmonary. In yet otherembodiments, the administration route is intrabronchial. In yet otherembodiments, the administration route is selected from the groupconsisting of oral, parenteral, nasal, inhalational, intratracheal,intrapulmonary, and intrabronchial. In yet other embodiments, theadministration route is selected from the group consisting of nasal,inhalational, intratracheal, intrapulmonary, and intrabronchial. In yetother embodiments, the administration is done using a nebulizer. In yetother embodiments, the acute lung injury is acute respiratory distresssyndrome.

In certain embodiments, the compositions of the invention areadministered to the subject about three times a day, about twice a day,about once a day, about every other day, about every third day, aboutevery fourth day, about every fifth day, about every sixth day and/orabout once a week.

In certain embodiments, the dose of pazopanib, or a salt or solvatethereof, required to treat acute lung injury in a subject using a routeof administration selected from the group consisting of nasal,inhalational, intratracheal, intrapulmonary, intrabronchial, andinhalation, is lower than the dose of pazopanib, or a salt or solvatethereof, required to treat cancer (such as but not limited to advancedrenal cell carcinoma) in a subject orally. In other embodiments, thedose used within the methods of the invention is about 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45,1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95 or 1:100 thatof the oral dose required to treat cancer, in terms of mass ofpazopanib, or a salt or solvate thereof, per subject's weight. In yetother embodiments, the dose of drug is about 5-200 mg/day.

In certain embodiments, administration of the compound and/orcomposition to the subject does not cause significant adverse reactions,side effects and/or toxicities that are associated with systemicadministration of the compound and/or composition. Non-limiting examplesof adverse reactions, side effects and/or toxicities include, but arenot limited to hepatotoxicity (which may be evidenced and/or detected byincreases in serum transaminase levels and bilirubin), prolonged QTintervals and torsades de pointes, hemorrhagic events, decrease orhampering of coagulation, arterial thrombotic events, gastrointestinalperforation or fistula, hypertension, hypothyroidism, proteinuria,diarrhea, hair color changes (depigmentation), nausea, anorexia, andvomiting.

In certain embodiments, the subject is undergoing treatment in anintensive care unit (ICU). In other embodiments, the subject isundergoing treatment in an emergency room (ER). In yet otherembodiments, the subject is on a ventilator.

In certain embodiments, the subject is further administered at least oneadditional agent that treats, prevents or reduces the symptoms of thelung fibrosis and/or acute lung injury.

In certain embodiments, the subject is a mammal. In other embodiments,the mammal is a human.

The invention further provides a method of evaluating efficacy of a drugin treating ALI.

In certain embodiments, the method comprises contacting a neutrophilwith the drug and measuring neutrophil ROS production levels after thecontacting. If the neutrophil ROS production levels increase after thecontacting, the drug is efficacious in treating ALI.

The invention further provides a method of evaluating efficacy of a drugin treating a subject suffering from ALI. In certain embodiments, themethod comprises measuring neutrophil ROS production levels in thesubject after being administered the drug. If the neutrophil ROSproduction levels in the subject after being administered the drug arehigher than the neutrophil ROS production levels in the subject beforebeing administered the drug, the drug is efficacious in treating ALI inthe subject.

Kits

The invention includes a kit comprising pazopanib, or a salt or solvatethereof, an applicator, and an instructional material for use thereof.The instructional material included in the kit comprises instructionsfor preventing or treating lung fibrosis and/or acute lung injury, orany other disease or disorder contemplated within the invention. Theinstructional material recites the amount of, and frequency with which,the pazopanib, or a salt or solvate thereof, should be administered tothe subject. In other embodiments, the kit further comprises at leastone additional agent that treats, prevents or reduces the symptoms oflung fibrosis and/or acute lung injury.

Combination Therapies

In certain embodiments, the compounds of the invention are useful in themethods of the invention in combination with at least one additionalcompound and/or therapy useful for treating or preventing lung fibrosisand/or acute lung injury. This additional compound may comprisecompounds identified herein or compounds, e.g., commercially availablecompounds, known to treat, prevent or reduce the symptoms of lungfibrosis and/or acute lung injury.

Non-limiting examples of additional therapies contemplated within theinvention include low tidal volume ventilation, which is a standard caretherapy for ALI/ARDS.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22:27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the onset of a disease or disorder contemplatedin the invention. Further, several divided dosages, as well as staggereddosages may be administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection. Further, the dosagesof the therapeutic formulations may be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder contemplated in the invention. Aneffective amount of the therapeutic compound necessary to achieve atherapeutic effect may vary according to factors such as the state ofthe disease or disorder in the patient; the age, sex, and weight of thepatient; and the ability of the therapeutic compound to treat a diseaseor disorder contemplated in the invention. Dosage regimens may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. A non-limiting example of an effective dose range for atherapeutic compound of the invention is from about 0.01 and 5,000 mg/kgof body weight/per day. One of ordinary skill in the art would be ableto study the relevant factors and make the determination regarding theeffective amount of the therapeutic compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The therapeutically effective amount or dose of a compound of thepresent invention depends on the age, sex and weight of the patient, thecurrent medical condition of the patient and the progression of adisease or disorder contemplated in the invention.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

A suitable dose of a compound of the present invention may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In certain embodiments, the compositions of the invention areadministered to the patient in dosages that range from one to five timesper day or more. In another embodiment, the compositions of theinvention are administered to the patient in range of dosages thatinclude, but are not limited to, once every day, every two, days, everythree days to once a week, and once every two weeks. It is readilyapparent to one skilled in the art that the frequency of administrationof the various combination compositions of the invention varies fromindividual to individual depending on many factors including, but notlimited to, age, disease or disorder to be treated, gender, overallhealth, and other factors. Thus, the invention should not be construedto be limited to any particular dosage regime and the precise dosage andcomposition to be administered to any patient is determined by theattending physical taking all other factors about the patient intoaccount.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of thedisease or disorder, to a level at which the improved disease isretained. In certain embodiments, patients require intermittenttreatment on a long-term basis upon any recurrence of symptoms and/orinfection.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withminimal toxicity. The dosage optionally varies within this rangedepending upon the dosage form employed and the route of administrationutilized.

In certain embodiments, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Incertain embodiments, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof a disease or disorder contemplated in the invention.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for any suitable mode of administration, known tothe art. The pharmaceutical preparations may be sterilized and ifdesired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., analgesic agents.

Routes of administration of any of the compositions of the inventioninclude nasal, inhalational, intratracheal, intrapulmonary, andintrabronchial.

Suitable compositions and dosage forms include, for example,dispersions, suspensions, solutions, syrups, granules, beads, powders,pellets, liquid sprays for nasal or oral administration, dry powder oraerosolized formulations for inhalation, and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to form amaterial that is suitable to administration to a subject. Each of theseformulations may further comprise one or more of dispersing or wettingagent, a suspending agent, and a preservative. Additional excipients,such as fillers and sweetening, flavoring, or coloring agents, may alsobe included in these formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles thatcomprise the active ingredient and have a diameter in the range fromabout 0.5 to about 7 nanometers, and preferably from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The pharmaceutical composition of the invention may be delivered usingan inhalator such as those recited in U.S. Pat. No. 8,333,192 B2, whichis incorporated herein by reference in its entirety.

In certain embodiments, the composition of the invention comprises astable dry powder blend containing levothyroxine sodium hydrate; lactoseparticles, comprising lactose H₂O, gelatin and starch maize; sodiumstarch glycolate; magnesium stearate; and talc silicified, comprisingtalc purified and colloidal silicon dioxide. In other embodiments, thedry powder comprises levothyroxine sodium is in an amount 4 to 0.02 mgper 100 mg of the dry powder. In yet other embodiments, the dry powdercomprises lactose in an amount higher than 90 mg per 100 mg of the drypowder preparation. In yet other embodiments, the dry powder compriseslactose particles consisting of lactose H₂O, gelatin and starch maize,wherein the ratio by weight-mg of: “lactose H₂O”:“gelatin”:“starchmaize” is 55-75:0.20-0.80:20-40. In yet other embodiments, the drypowder comprises sodium starch glycolate in an amount of 4-8 mg per 100mg of dry powder. In yet other embodiments, the dry powder comprisesmagnesium stearate in an amount of 0.5-2 mg per 100 mg of dry powder. Inyet other embodiments, the dry powder comprises talc silicified, in anamount of 2 mg per 100 mg of dry powder, wherein the talc silicifiedcomprises talc purified and colloidal silicon dioxide in an amount of0.667 mg of talc purified and 1.333 mg of colloidal silicon dioxide for2 mg of talc silicified. In yet other embodiments, the blend furthercomprises a lake. In yet other embodiments, the dry powder comprisessodium starch glycolate in an amount of 5-6 mg per 100 mg of dry powder.In yet other embodiments, the dry powder comprises magnesium stearate inan amount of 1 mg per 100 mg of dry powder.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material that provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In certain embodiments of the invention, the compounds of the inventionare administered to a patient, alone or in combination with anotherpharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that may,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction and/or treatmentconditions with art-recognized alternatives and using no more thanroutine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Methods:

Materials

The following reagents were purchased from Sigma:N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), Phorbol12-myristate 13-acetate (PMA), Lipopolysaccharide (LPS), ButylatedHydroxyanisole (BHA), Lysolecithin, Paraformaldehyde (PFA), FITCAlbumin, Horse Reddish Peroxidase (HRP), and Isoluminol. Percoll waspurchased from GE Healthcare (Uppsala, Sweden), Bovine Serum Albumin(BSA) was purchased from American Bio (Natick, Mass.), GMCSF waspurchased from Peprotech, Lipofectamine kit and Cell trace dyes werepurchased from Thermo Fisher. The following material was purchased fromGIBCO: Dulbecco's Modified Eagle Medium (DMEM), Hanks Balanced SaltSolution (HMS), Phosphate Buffered Saline (PBS).

The commercial antibodies used in the study are: GST antibody (2624,Cell signaling), His antibody (2366, Cell Signaling), HA antibody(MMS-101R, Covance), Myc antibody (MMS-150R, Covance), anti-phospho-AKTantibody (4060 and 2965, Cell Signaling), anti-AKT antibody (9272, CellSignaling), anti-MEKK3 antibody (5727, Cell Signaling), anti-p47^(phox)antibody (17875, Santa Cruz), anti-CD31 antibody (102502, BioLegend),anti-α-smooth muscle actin antibody (ab8211, Abcam), and anti-β-actinantibody (4967, Cell Signaling). The rabbit polyclonal anti-S208p47^(phox) was acquired from Abiocode. Protein A/g PLUS-agarose beadswere purchased from Sant Cruz Biotechnology (Santa Cruz, Calif.). ELISAkits for cytokine measurements were purchased from eBioscience (SanDiego, Calif.). The cDNAs for MAP3K3 and p67^(phox) were acquired fromADDGENE, and cDNAs for p47^(phox) and gp91^(phox) from Open Biosystems.

Mice

The MEKK2^(−/−) mice were previously described in Guo, et al., 2002,Mol. Cell Biol. 22:5761-5768, whereas the MEKK3″ mice were described inWang, et al., 2009, J. Immunol. 182:3597-3608. Both MEKK2^(−/−) andMEKK3^(fl/fl) are in C57B1 background. Myeloid cell specific MEKK3 KOmice, MEKK3^(m/m), were generated by intercrossing MEKK3^(fl/fl) micewith the B6.129-Lyzs^(tml(cre)Ifo)/J mice from Jackson Lab. The doubleknockout (DKO) mice, MEKK2^(−/−)MEKK3^(m/m), were generated byintercrossing MEKK2^(−/−) mice with MEKK3^(m/m) mice. Wild type (WT)mice, C57BL6, were purchased from Taconic laboratories (Germantown,N.Y.). The BHA (W218308, Sigma-Aldrich)-containing chow (0.75% w/w BHA)was custom-made by Harlan Laboratories from 2018S diet and sterilized byirradiation.

Neutrophil Preparation and Transfection

Mice were euthanized in a CO₂ chamber according to approved protocol,bone marrow was harvested from long bones of the mice, red blood cells(RBCs) were lysed with ACK buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 127 μMEDTA), the cells were layered on a discontinuous Percoll gradientcomposed of 81%, 62% and 45% Percoll layers, and the cells were isolatedfrom the interphase between 81% and 62% Percoll layers. Cells werewashed in MSS and used for various assays.

For neutrophil transfection, neutrophils (3×10⁶ cells/100 μl) and up to1.6 μg of DNA were suspended in the supplied nucleofection solution andelectroporated in Nucleofector device 2b (Lonza, Switzerland). Thesamples were then cultured overnight in the medium (RPMI 1640, 10% FBS(V/V), GMCSF 25 ng/ml) at 37° C. in humidified air with 5% CO₂.

Dunn Chamber Chemotaxis Assay

WT (stained with Cell Trace Calcein Red-Orange dye) and DKO neutrophils(1.25×10⁶ cells/ml) were suspended in an assay buffer (0.25% BSA in MSSwith Ca²⁺ and Mg²⁺), and vice versa. An aliquot of cells was thenallowed to adhere for 15 minutes on fibrinogen coated coverslips, thecoverslip was inverted on the Dunn Chamber with assay buffer in theinner well and fMLP (10 μM) in the outer well, and time lapse imageswere recorded at 30 sec intervals for 30 minutes under Olympus BX61microscope. The cellular tracks were analyzed as reported in Konstandin,et al., 2006, J. Immunol. Meth. 310:67-77.

Integrin Expression Assay

Bone marrow-derived neutrophils were resuspended in flow cytometrybuffer (PBS with 1% BSA), stimulated with fMLP (5 μM) for indicateddurations, fixed with 4% PFA and then stained with FITC labeled antiLFA-1 or anti Mac-1. Samples were analyzed by BD LSR II flow cytometer.

ICAM-1 Binding Assay

The assay was carried out as described in Wang, et al., 2008, J. Clin.Invest. 118:195-204. The ICAM-1-Fc-F(ab′)2 complexes were generated byincubating Cy5-conjugated AffiniPure goat anti-human Fcγfragment-specific IgG F(ab′)2 fragments (Jackson Immunobiology) andICAM-1-Fc (100 μg/ml, R&D) at 4° C. for 30 min in PBS. Neutrophils,which were resuspended at 0.5×10⁶ cells/ml in PBS containing 0.5% BSA,0.5 mM Mg²⁺ and 0.9 mM Ca²⁺, were mixed with the ICAM-1-Fc-F(ab′)2complexes in the presence or absence of fMLP for durations specified inthe figure legends. The reactions were terminated by adding 4%paraformaldehyde. After 5 min, fixation was stopped by adding 3-mlice-cold FACS buffer. Cells were pelleted, resuspended in 300 μl of FACSbuffer, and analyzed on a flow cytometer.

Neutrophil Infiltration into Inflamed Peritonea and Flow ChamberAdhesion Assay

For the peritonitis infiltration model, purified wild type and mutantneutrophils were labeled with 2.5 μM CFSE [5-(and -6)-carboxyfluoresceindiacetate succinimidyl esters] and 2.5 μM Far-Red DDAO SE, respectively,and vice versa. The WT and mutant cells with different fluorescencelabels were mixed at a 1:1 ratio and injected into retro-orbital venoussinus of wildtype littermates, which were injected with 2 ml of 3%Thioglycolate (TG) two hours earlier. The mice were euthanized one andhalf hour later. Cells in their peritonea were collected and analyzed bycell counting and flow cytometry. The data presented are the combinationof the experiments with reciprocal fluorescence labeling.

To examine neutrophil adherence to endothelial cells under shear stress,mouse endothelial cells (Wang, et al., 2008, J. Clin. Invest.118:195-204) were cultured to confluency on 10 μg/ml fibronectin coatedcoverslips and treated with 50 ng/ml TNFα for 4 hours. The coverslipscontaining the endothelial cell layer were washed with PBS and placed ina flow chamber apparatus (GlycoTech). The WT and mutant cells labeleddifferent fluorescence labels as described elsewhere herein were mixedat a 1:1 ratio and flowed into the chamber at a shear flow rate of 1dyn/cm². The adherent cells were then examined and counted under afluorescence microscope.

ROS Release Assay

Neutrophils were suspended in a reaction mixture (0.25% BSA in MSS withCa²⁺ and Mg²⁺, 10 mM Isoluminol, 100μ/ml HRP), distributed cells in thewell of a 96 well plate, and stimulated with fMLP (10 μM).Isoluminol-enhanced chemiluminescence was recorded continuously in aplate reader (Perkin Elmer). For restituted ROS production system inCOS-7 cells, PMA (2 μM) was used for stimulation.

Neutrophil Degranulation Assay

One million neutrophils were incubated with 10 μM CB for 5 min at 37° C.prior to stimulation with fMLP (500 nM) for another 10 min. The reactionwas stopped by being placed on ice, and the suspension was centrifugedat 500×g for 5 min at 4° C. Supernatants were assayed for MPO and MMPcontents using the EnzChek Myeloperoxidase Activity Assay Kit andEnzChek Gelatinase/Collagenase Assay kit (Life Technologies, GrandIsland, N.Y.), respectively (Li, et al., 2009, Blood 113:4930-4941; Lee,et al., 2007, Am. J. Physiol. Lung Cell. Mol. Physiol. 292:L799-812).

LPS-Induced Lung Injury

Mice were anesthetized with ketamine/Xylazine (1 gm/kg and 100 mg/kg)and were allowed to inhaled 50 μl of LPS (1 mg/ml) placed as droplets onnares. Mice postures were maintained upright. Twenty-two hours after theinduction of injury, 100 μl of FITC-labeled albumin (10 mg/ml) wereinjected via retro-orbital vein, and 24 hours after the induction ofinjury, mice were euthanized by exsanguination. To obtainbronchoalveolar lavage fluid, 1 ml of PBS was instilled into lungs andretrieved a via a tracheal catheter. In some experiments, mice werefirst fed with antioxidant BHA in food (Harlan Laboratory Services) for7 days before the induction of lung injury.

Acid Aspiration-Induced Lung Injury

Mice were anaesthetized by ketamine/Xylazine (1 gm/kg and 100 mg/kg) andwere suspended vertically from their incisors on a custom-made mount fororotracheal instillation. A 22G catheter (Jelco, Smiths Medical) wasguided 1.5 cm below the vocal cords, and 2.5 μl/g of 0.05 M HCl wasinstilled. Two hours after the induction of injury, 100 μl ofFITC-labeled albumin (10 mg/ml) was injected via retro-orbital vein.Measurements were made 6 hours after the induction of injury. Controlanimals received saline instead of HCl in the same manner. In survivalexperiments, mice received 2.5 μl/g of 0.1 M HCl orotracheally and theobservation period was extended up to 30 h. To examine pharmacologicalintervention, MEKK2/3 inhibitor pazopanib were used 1 h after HClinstillation.

GST Pulldown Assay

Recombinant proteins were expressed in E. coli and purified by affinitychromatography. The proteins were then incubated in 200 μl of thebinding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton, 0.12% SDS, 1mM dithiothreitol, 10% glycerol, lx protease inhibitor cocktail) at 4°C. overnight on a shaker. Next morning, glutathione beads were added tothe protein mixture for additional 2 h. After extensive washes, proteinson the beads were resolved by SDS/PAGE and detected by Western Blot.

MAP3K3 Kinase Assay

In 50 μl reaction buffer (100 mM Tris-HCl pH 7.4, 50 mM EGTA, 100 mMMgCl₂), 100 ng of recombinant MAP3K3 were incubated withimmune-precipitated substrate proteins, [γ-³³P]-ATP (10 μCi), and coldATP (50 μM) at 37° C. for 30 minutes. The reaction was stopped by addingthe SDS loading buffer. The samples were boiled for 5 minutes. Theproteins were separated by SDS-PAGE, and were visualized and quantifiedby a phosphoimager.

Adoptive Bone Marrow Transfer

Marrows were harvested from the long bones of WT and DKO donor mice,RBCs were lysed with ACK buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 127 μMEDTA), cells were suspended in sterile normal saline and intravenouslyinjected into irradiated (9.5 Grays, γ radiation) recipient WT mice(5×10⁶ cells/mouse). The mice were provided with autoclaved food andwater containing Sulfatrim (48 mg/ml) for four weeks. These mice (HS-DKOand WT control) were used for experimental purposes 8 weeks aftertransplantation.

Human Neutrophils

Buffy coat of human blood samples were subjected to neutrophilenrichment using the EasySep Human Neutrophil Enrichment Kit (StemcellTechnologies) according to manufacturer's protocol. Briefly, thedepletion antibody cocktail was mixed with the buffy coat followed byincubation with magnetic particles. The EasySep Magnet was then used toimmobilize unwanted cells as the label-free neutrophils were poured intoanother conical tube. Enriched neutrophils were pelleted and resuspendedin an assay buffer (Hanks buffer with Ca²⁺ and Mg²⁺, 0.25% BSA) for ROSproduction assay.

Bi-Layer Co-Culture of Neutrophils with Endothelial Cells

Mouse endothelial cell (MEFC; Paik, et al., 2004, Genes Dev.18:2392-2403) were first plated on the outside of the polycarbonatemembrane (25,000 cells/cm²) of the Transwell inserts (24-well type,0.4-μm pore size, Corning, Inc. 353095), and placed upside down in thewells of the culture plate. After the endothelial cells had adhered, theTranswell inserts were inverted and reinserted into the wells of theplate. The medium was replaced 24 h after seeding with serum-freemedium. SOD (60 U/ml), catalase (100 U/ml) or mock were added to thelower chambers 2 h later for 30 min. Mouse neutrophils stimulated with 5μM fMLP were then plated on the top surface of the insert (6×10⁶cells/cm²) for 30 min. At the end of the incubation period, neutrophilson the top side of the inserts were scraped, and endothelial cells onthe other side of the inserts were lysed with SDS-PAGE sample buffer forWestern analysis.

Trans-Endothelial Electrical Resistance (TEER) Measurement

ECIS 8W10E+ arrays (Applied BioPhysics) were coated with 10 μg/ml ofpoly-D-lysine (PDL) and washed with sterile water. Complete EBM-2 media(300 μl) was added to each well for a quick impedance background check.Subsequently, immortalized mouse pulmonary endothelial cells (Murata, etal., 2007, J. Biol. Chem. 282:16631-16643) were seeded in a density of60,000 cells/well in 300 μl EBM-2 medium in the coated arrays andincubated them at 37° C. in a CO₂ incubator. Electrical resistance ofthe cell layer was recorded continuously on an ECIS system (AppliedBioPhysics) until a stable resistance of approximately 600-700 ohms wasachieved, after which media were removed from wells and replaced with100 μl of assay buffer (Hanks buffer with Ca²⁺ and Mg²⁺, 0.25% BSA).Cells were allowed to re-equilibrate at 37° C. for 2 hours, before 1 μlof SOD (60 U/ml), catalase (100 U/ml) or mock were added to wells for 30min followed by addition of 50 μl of mouse neutrophils in assay buffercontaining 5 μM fMLP. Data were collected real-time throughout theexperiment. All ECIS measurements were analyzed at an AC frequency of 4kHz, which was identified as the most sensitive frequency for this celltype by frequency scans along an entire frequency range (1 kHz-64 kHz).The TEER values were normalized against those co-cultured with WTneutrophils treated with mock.

Statistical Methods

Data were analyzed with Prism software. For two samples, t test wasused; for multiple samples, ANOVA was used with p values set at <0.05 asbeing significant.

Example 1: MAP3K2/3-Deficiency Ameliorates LPS-Induced Lung Injury

Gene expression analysis indicates that the MAP3K3 gene is specificallyexpressed in human myeloid cells (www dot biogps dot org). In addition,its expression is down-regulated in neutrophils from the lung exudatesof human subjects inhaled with endotoxin. Because of the importance ofneutrophils in acute lung injury, the role of MAP3K3 in myeloid cellfunctions and acute lung injury was investigated using a mouse acutelung injury (ALI) model.

In mice, the Map3k3 gene is expressed abundantly in varioushematopoietic cells with its expression being highest in myeloid cells(www dot immgen dot org). Myeloid-specific knockout (KO) of Map3k3 wasgenerated by crossing Map3k3^(fl/fl) and lysozyme-Cre mice. However,significant neutrophil or lung injury phenotypes were not observed withMap3k3-deficiency. Without wishing to be limited by any theory, MAP3K3function may be compensated by its close homolog MAP3K2, which is alsoexpressed in mouse myeloid cells (www dot immgen dot org) and, likeMAP3K3, could be readily detected in neutrophils by Western analysis(FIG. 1A).

Thus, both kinases were inactivated, and subsequently a global MAP3K2knockout (KO) and myeloid-specific MAP3K3 KO mouse line (DKO) wasgenerated by crossing the Map3k2^(−/−) mice with myeloid-specificMap3k3^(−/−) mice. To limit contributions of MAP3K2 fromnon-hematopoietic cells, adoptive transfer of the DKO bone marrow (BM)to lethally irradiated wildtype (WT) recipient mice was performed. Theresultant mice are designated as HS-DKO. Western analysis shows the lackof the MAP3K2 and MAP3K3 proteins in the neutrophils isolated from theHS-DKO mice (FIG. 1A).

The HS-DKO mice were subjected to LPS-induced lung injury. This murinemodel recapitulates the hallmarks of human ALI including neutrophilicinflux into the alveolar space, pulmonary edema, increased lungpermeability (FIG. 8A), and high mortality. When the HS-DKO mice andcontrol WT mice, which received WT BM transfer, were treated with LPSvia nasal instillation, the HS-DKO mice sustained significantly reducedlung injury compared to the wildtype (WT) control mice, evidenced byreduced permeability, edema and alveolar wall thickening (FIGS. 1B-1D).The HS-DKO mice also showed reduced mortality compared to the WT controlmice (FIG. 1E). The same results were also observed when HCl-ALI modelwas used; the lack of MEKK2/3 reduced lung permeability and damage andextended survival (FIGS. 1F-1H). There was no significant difference inthe numbers of myeloid cells in the bronchoalveolar lavage fluid (BALF)between the HS-DKO and WT control mice (FIGS. 8B-8D). In addition, therewere no significant differences in the contents of TNFα, IL-1β or IL-6in BALFs (FIG. 8E). No differences in myeloid infiltration or IL-1blevel in BALF were observed when the HCl model was used (FIG. 8F-J).Together, these results suggest that the lack of MAP3K3 in myeloid cellsand MAP3K2 in hematopoietic cells largely affects pulmonary permeabilityrather than myeloid infiltration or cytokine production in injuredlungs.

Example 2: MAP3K2/3-Deficiency Specifically Alters Neutrophil ROSRelease

Consistent with the lack of an effect on neutrophil infiltration intoBALF by MAP3K2/3-deficiency, the deficiency did not affect neutrophilchemotaxis in in vitro (FIGS. 2A-2D). Neither did it affect neutrophiladhesion to endothelial cells (FIG. 2E), nor expression or activation ofβ2 integrins (FIGS. 2F-2H). There was also no difference between WT orMAP3K2/3-deficient neutrophils in infiltration into inflamed peritonea,a model for testing neutrophil infiltration in vivo (FIG. 2I). Inaddition, MAP3K2/3-deficiency did not alter neutrophil degranulation(FIGS. 2J-2K). However, the MAP3K2/3-deficiency led to increasedproduction of ROS from neutrophils upon stimulation (FIG. 3A).Expression of WT, but not kinase-dead, MAP3K3 in the MAP3K2/3-deficientneutrophils could suppress the ROS release, confirming the involvementof MAP3K3 in regulation of ROS release (FIG. 3B). This result alsoindicates that MAP3K3 regulated-ROS release is dependent on its kinaseactivity. When the mice were fed with butylated hydroxyanisole (BHA), aROS scavenger and subjected to LPS-induced injury, the differencebetween HS-DKO and WT control mice in permeability and edema dissipated(FIGS. 3C-3E), suggesting that the protective role ofMAP3K2/3-deficiency depends on ROS.

Effects of individual MAP3K2 and MAP3K3 KO on ROS release fromneutrophils were also examined. MAP3K3 KO showed a trend of increases inROS release, whereas MAP3K2 KO had no significant effect (FIGS. 9A-9B).These results confirm that these two kinases are indeed functionallyredundant.

Example 3: MAP3K3 Phosphorylates p47^(phox) to Inhibit ROS Production

Given that the phagocytic NADPH oxidase is the major source of ROSproduction in neutrophils, it was tested if MAP3K3 acted through thisenzyme complex. It was first investigated if the NADPH oxidase can be asubstrate of MAP3K3. Recombinant proteins of MAP3K3 and several subunitsof the NADPH oxidase were produced and in vitro kinase assays werecarried out. Only p47^(phox), but not p22^(phox) or p67^(phox), could bephosphorylated by MAP3K3 (FIG. 4A). Though the phosphorylation siteconsensus sequence for MAP3K3 is unknown, the p47^(phox) sequence wasanalyzed using the program Scansite run with reported peptide array datafor the related kinase MAP3K5 to identify likely sites ofphosphorylation. This analysis predicted Ser-208 as the best scoringsite among those previously observed (Obenauer, et al., 2003, NucleicAcids Res. 31:3635-3641). When this site was mutated, MAP3K3-mediatedphosphorylation was significantly reduced (FIG. 4B), indicating thatthis residue can indeed be phosphorylated by MAP3K3.

Next effects of this phosphorylation on the activity of the NDAPHoxidase were evaluated. The NADPH oxidase activity in COS-7 cells wasreconstituted by expressing the NADPH oxidase subunits p47^(phox),p67^(phox), p40^(phox), NOX2, and p22^(phox) (Price, et al., 2002, Blood99:2653-2661). These proteins are either not or insufficiently expressedin COS-7 cells. Upon addition of PMA, production of ROS could bedetected from the reconstituted COS-7 cells, and this ROS production iscompletely dependent on the exogenous expression of p47^(phox) (FIGS.10A-10B). Expression of WT MAP3K3, but not its kinase dead mutant,inhibited ROS production in this system (FIG. 10C). Thus, a ROSproduction system that can be inhibited by MAP3K3 was reconstituted,similar to what happens in neutrophils. When the phospho-mimeticp47^(phox) S208E mutant was used instead of WT in this reconstitutedsystem, there was very low ROS production in comparison to WT p47^(phox)(FIG. 4C). The non-phosphorylatable S208A p47^(phox) mutant, bycontrast, showed similar activity in the ROS reconstitution assay to theWT p47^(phox) (FIG. 4C). Moreover, expression of MAP3K3 inhibited ROSproduction in cells expressing the WT p47^(phox), but not thoseexpressing the non-phosphorylatable S208A p47^(phox) (FIG. 4D). Theseresults together indicate that MAP3K3-mediated phosphorylation ofp47^(phox) at S208 inhibits the NADPH oxidase activity.

Because Ser-208 is located between two SH3 domains of p47^(phox), whichwere involved in the interaction with p22^(phox) during activation ofthe NADPH oxidase complex (FIG. 10D), in certain non-limitingembodiments the phosphorylation at Ser-208 can interfere with thisinteraction, a critical step in NADPH oxidase activation. Indeed, thephosphomimetic Ser-208 to Glu mutation abolished the interaction ofp47^(phox) with p22^(phox) in a co-immunoprecipitation assay (FIG. 4E).

Example 4: Ser-208 of p47^(phox) is Phosphorylated in Neutrophils

To detect if p47^(phox) is phosphorylated in neutrophils by MAP3K2/3, anantibody specific for phosphorylated Ser-208 of p47^(phox) wasgenerated. Validation assay indicates that the antibody is largelyspecific for Ser-208-phosphorylated p47^(phox), because Ser-208 mutationto alanine markedly diminishes the detection by the antibody (FIGS.11A-11G). Using the antibody, we detected time-dependent increases inp47^(phox) phosphorylation at Ser-208 (FIG. 4F). The time course offMLP-stimulated increases in p47phox phosphorylation coincides with thatof AKT phosphorylation at Ser-473 (FIG. 4F). In addition, increases inabundance of the MAP3K3 protein were observed, which may reflect itsactivation. The fMLP-induced increase in p47phox phosphorylationdetected by this antibody was not observed in neutrophils lackingMAP3K2/3 (FIG. 4G), suggesting that fMLP induces the phosphorylation ofp47phox at Ser-208 via MAP3K2/3. Without wishing to be limited by anytheory, the bands detected in the DKO neutrophils by the antibody mayreflect the detection of either non-phosphorylated p47phox by theantibody or basal phosphorylation of Ser-208 by other protein kinases.

In vitro kinase assays were performed to determine the IC₅₀ for theinhibition of MEKK2 and 3 by pazopanib. Pazopanib inhibited MEKK3 withan IC₅₀ about 10 nM, whereas inhibiting MEKK2 with an IC₅₀ of 20 nM(FIGS. 4H-41). These values are much lower than the IC₅₀ (>1 μM) for theonly published MEKK2/3 substrate, MEK5 (FIGS. 4J-4K).

Example 5: Pazopanib Inhibits MAP3K2/3 and Reduces Lung Injury

Pazopanib is a FDA-approved drug for targeted cancer therapy. Itinhibits a number of receptor tyrosine kinases including receptors forVEGF, FGF, PDGF and SCF (Keisner & Shah, 2011, Drugs 71:443-454). Italso inhibits MAP3K2 with a potency comparable to its originallyintended targets (Ahmad, et al., 2013, J. Biomol. Screen. 18:388-399).Pazopanib was tested in neutrophils and found to inhibit p47^(phox)phosphorylation at Ser-208 detected by the phospho-specific antibody(FIG. 5A). Pazopanib also abrogated increase in MAP3K3 protein contentinduced by fMLP (FIG. 5A). Because MAP3K3 activates viaautophosphorylation, this result is consistent with the idea that MAP3K3stabilization may be a result of its activation and further confirmsthat pazopanib acts through MAP3Ks. Treatment of WT (FIG. 5B), but notMAP3K2/3-deficient (FIG. 5C), mouse neutrophils with pazopanib led toincreases in the ROS production, indicating that pazopanib increases ROSproduction via MAP3K2/3. In addition, pazopanib increased ROS productionfrom human neutrophils (FIGS. 12A-12B). The WT mice that were subjectedto LPS-induced injury were fed with pazopanib. Similar to MAP3K2/3HS-DKO, pazopanib treatment showed reduced pulmonary permeability (FIG.5D), alveolar wall thickening, and edema (FIG. 5E). More importantly,pazopanib treatment reduced mortality in a prophylactic (FIG. 5F) ortherapeutic (FIG. 12E) modality. For the prophylactic treatment,pazopanib was given two days before the lung injury, whereas the drugwas given 24 hour after the injury.

Another model of acute lung injury induced by acid aspiration was used.Acid aspiration-induced ALI, also known as aspiration pneumonitis,results from pulmonary aspiration of the acid content of the stomach.This frequently occurs to patients with disturbed consciousness (e.g.,drug overdose, seizures, cerebrovascular accident, sedation, anestheticprocedures) and is accounts up to 30% of all deaths associated withanesthesia. In this aspiration-induced ALI model, pazopanib treatmentdecreased pulmonary permeability (FIG. 12F), alveolar wall thickening,and edema (FIG. 12G). In addition, the treatment significantly extendedthe survival (FIG. 12H). Thus, these data together clearly demonstratethat pazopanib inhibit MAP3K2/3 and provide effective treatment in twodifferent ALI models.

The preventative effect of pazopanib in the HCl model was also tested.Pazopanib was effective in reduction of permeability and extendingsurvival (FIGS. 12I-12J).

Another tyrosine kinase inhibitor, imatinib, was also tested, and foundto be ineffective in reducing lung permeability in the HCl-induced lunginjury (FIG. 17). This result shows that the presently describedbeneficial effect of pazopanib is not shared by other tyrosine kinaseinhibitors.

Example 6: Pazopanib Ameliorates Lung Injury Via MEKK2/3, p4′7, but notVEGFR

FIG. 12C-12D illustrates the finding that pazopanib ameliorates lunginjury via MEKK2/3, as the drug had no effects on lung injury in micelacking these kinases. In addition, pazopanib showed no effects in micelacking p47^(phox) (FIGS. 16C & 16E), the key subunit that produces ROSin neutrophils. The fact that mice lacking p47^(phox) are moresusceptible to the lung injury (FIGS. 16A-16B & 16D) is consistent withthe finding that neutrophil ROS is protective in lung injury.

Pazopanib also inhibits VEGFR. The effect of a neutralizing anti-VEGFRantibody was tested in the present model, and showed no effect insurvival of mice subjected to lung injury (FIG. 15). Thus, pazopanib'sinhibition of VEGFR does not play an important role in ameliorating lunginjury.

Example 7: MAPK2/3 Inhibition Increases Lung AKT Activation

AKT signaling has a protective role in a murine model of ALI bypreventing capillary leakage and clearing alveolar fluid. Moreover, ROSstimulates AKT activation in endothelial cells to strengthen vesselbarrier integrity. AKT phosphorylation at Ser-473 was thus examined inLPS-treated lung samples, elevated AKT phosphorylation in the HS-DKOsamples were found as compared to the controls (FIG. 13A). Because therewas no difference in AKT phosphorylation between WT and DKO neutrophils(FIG. 5A), without wishing to be limited by any theory, the differencein AKT phosphorylation observed in the lung samples might be due to thedifferences in non-hematopoietic lung cells. Immunofluorescence of lungsections from LPS-treated mice showed higher levels of AKTphosphorylation in HS-DKO samples in pulmonary vessels and capillariesmarked by CD31 staining (FIG. 6 (Panels A-B) & FIG. 13B) and vascularsmooth muscle cells marked by smooth muscle actin staining (FIG. 6(Panels C-D) & FIG. 13C). By contrast, the phospho-AKT staining ofbronchial epithelial and smooth muscle cells was comparable between theWT and HS-DKO samples (FIG. 6 (Panels A-B) & FIGS. 13B-13C). Inaddition, pazopanib treatment recapitulates HS-DKO's effects on AKTphosphorylation; the inhibitor increased AKT phosphorylation inLPS-injured lungs (FIG. 13D). This effect of pazopanib depends on thepresence of MAP3K2 and 3, as the inhibitor had little effect on AKTphosphorylation in the HS-DKO lungs (FIG. 13E). Furthermore, treatmentof the mice with the AKT inhibitor MK-2206(8-[4-(1-Aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one)abrogated the effect of pazopanib on permeability (FIG. 13F).

The aforementioned data, together with the knowledge that ROS canstimulate AKT phosphorylation in endothelial cells, support anon-limiting model (FIG. 7A) to suggest that, during acute lung injury,MAP3K2/3-deficient neutrophils release more ROS, which augments AKTactivation in endothelial cells and vascular smooth muscle cells,leading to improved vascular integrity and reduced permeability. Tofurther test this non-limiting hypothesis, co-culture of WT and DKOneutrophils was performed with mouse lung endothelial cells. Mouseendothelial cells co-cultured with fMLP-activated DKO neutrophils hadelevated phospho-AKT compared to co-culture with activated WTneutrophils (FIG. 7B). This phospho-AKT elevation could be abrogated bythe presence of catalase, but not superoxidase dismutase (SOD) (FIG.7C). Catalase catalyzes the conversion of H₂O₂ to water, whereas SODconverts superoxide to H₂O₂. Moreover, co-culture of activated DKOneutrophils with mouse endothelial cells increased trans-endothelialelectrical resistance (PEER) over that of activated WT neutrophils, andthis difference in TEER could also be abrogated by the addition ofcatalase (FIG. 7D). Thus, these results together support the conclusionthat activated DKO neutrophils can elevate phospho-AKT and improveendothelial junction integrity in co-cultured endothelial cells via H₂O₂and is consistent with the non-limiting model described in FIG. 7A.

Knowing that AKT may regulate endothelial junction integrity viaactivation of RAC1 small GTPase, it was tested if H₂O₂ can activate RACin mouse endothelial cells. Indeed, H₂O₂ was found to activate RAC1 inthe endothelial cell. In addition, co-culture of neutrophils lackingMEKK2/3 led to greater RAC1 activation than WT neutrophils, suggestingMEKK2/3 KO neutrophils can cause hyperactivation of RAC1 in endothelialcells. These findings are consistent with the hypothesis depicted inFIG. 7A.

A long detrimental effect of ALI is fibrosis. It was thus tested ifpazopanib can inhibit lung fibrosis. A bleomycin-induced lung fibrosismodel was used in this study: Gan, et al., 2012, Nat. Cell Biol. 14:686.Pazopanib was found to inhibit lung fibrosis (FIG. 18), suggesting thatthe mechanisms of action of pazopanib in curbing ALI are multifaceted.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed:
 1. A method of treating or ameliorating acute lunginjury (ALI) or lung fibrosis in a subject, the method comprisingadministering to the subject a therapeutically effective amount ofpazopanib, or a salt or solvate thereof, using an administration routeselected from the group consisting of oral, parenteral, nasal,inhalational, intratracheal, intrapulmonary, and intrabronchial; whereinthe pazopanib, or a salt or solvate thereof, is the only therapeuticagent administered to the subject to treat or ameliorate the ALI or lungfibrosis.
 2. The method of claim 1, wherein the administration is doneusing a nebulizer.
 3. The method of claim 1, wherein the subject is inan intensive care unit (ICU) or emergency room (ER).
 4. The method ofclaim 1, wherein the acute lung injury is acute respiratory distresssyndrome (ARDS).
 5. The method of claim 1, wherein the subject isfurther subjected to low tidal volume ventilation.
 6. The method ofclaim 1, wherein the-administration route is intravenous.
 7. The methodof claim 1, wherein the pazopanib, or a salt or solvate thereof, isadministered to the subject at a frequency selected from the groupconsisting of about three times a day, about twice a day, about once aday, about every other day, about every third day, about every fourthday, about every fifth day, about every sixth day and about once a week.8. The method of claim 1, wherein the pazopanib, or a salt or solvatethereof, is formulated as a dry powder blend.
 9. The method of claim 1,wherein administration of the pazopanib, or a salt or solvate thereof,to the subject does not cause at least one significant adverse reaction,side effect or toxicity associated with oral/systemic administration ofthe pazopanib, or a salt or solvate thereof, to a subject suffering fromcancer.
 10. The method of claim 9, wherein the at least one adversereaction, side effect or toxicity is selected from the group consistingof hepatotoxicity, prolonged QT intervals and torsades de pointes,hemorrhagic event, decrease or hampering of coagulation, arterialthrombotic event, gastrointestinal perforation or fistula, hypertension,hypothyroidism, proteinuria, diarrhea, hair color changes, nausea,anorexia, and vomiting.
 11. The method of claim 1, wherein the subjectis dosed with an amount of pazopanib, or a salt or solvate thereof, thatis lower than the amount of pazopanib, or a salt or solvate thereof,with which a subject suffering from cancer is dosed orally/systemicallyfor cancer treatment.
 12. The method of claim 1, wherein the subject isa mammal.
 13. The method of claim 12, wherein the mammal is a human. 14.A kit comprising pazopanib, or a salt or solvate thereof, an applicator,and an instructional material for use thereof, wherein the instructionalmaterial comprises instructions for treating or ameliorating acute lunginjury or lung fibrosis in a subject, wherein the pazopanib, or a saltor solvate thereof, is the only therapeutic agent to be administered tothe subject to treat or ameliorate the ALI or lung fibrosis, using anadministration route selected from the group consisting of nasal,inhalational, intratracheal, intrapulmonary, and intrabronchial.
 15. Thekit of claim 14, wherein the pazopanib, or a salt or solvate thereof, isformulated as a dry powder blend.